Protein folding in the bacterial periplasm

Missiakas, Dominique; Raina, Satish

doi:10.1128/jb.179.8.2465-2471.1997

Cited by 240 publications

(192 citation statements)

References 87 publications

(111 reference statements)

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“…In fact, the effects of SDS may be envisioned as the initial step on the folding pathway of class 3 protein in which a partially unfolded intermediate inserts into the membrane and eventually assumes its final ␤-sheet oligomeric conformation. Several proteins have been proposed as possible in vivo chaperone-like molecules involved in the folding/assembly of outer membrane proteins (55). However, there are multiple lines of experimental evidence including those presented in our laboratory (7,22, present study) that porins undergo complete folding from denaturant solutions solely in the presence of amphiphilic molecules such as detergents, thereby precluding assistance from molecular chaperones.…”

Section: Sds-induced Conformational Changes Of Classmentioning

confidence: 41%

Characterization of the Structure, Function, and Conformational Stability of PorB Class 3 Protein from Neisseria meningitidis

Minetti¹,

Blake²,

Remeta³

1998

Journal of Biological Chemistry

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PorB proteins constitute the vast majority of channels in neisserial outer membranes and can be subdivided within meningococcal strains into two distinct and mutually exclusive families that are designated as class 2 and class 3 proteins. We recently characterized the functional activity and conformational stability of a PorB class 2 protein from Neisseria meningitidis (Minetti, C. A. S. A., Tai, J. Y., Blake, M. S., Pullen, J. K., Liang, S. M., and Remeta, D. P. (1997) J. Biol. Chem. 272, 10710 -10720). To evaluate the structure-function relatedness among the PorB proteins, we have employed a combination of electrophoretic and spectroscopic techniques to assess the conformational stability of zwittergent-solubilized class 3 trimers. The functional, physicochemical, and structural properties of the meningococcal class 2 and class 3 proteins are comparable with the notable exception that the latter exhibits a significantly higher susceptibility to SDS. The SDS-induced dissociation and partial unfolding of PorB class 3 is characterized by a single two-state transition with a midpoint at 0.35% SDS. The native trimeric assembly dissociates reversibly, forming partially folded monomers that retain the characteristic ␤-sheet content of the transmembrane domain with a concomitant increase in random coil structure arising from unfolding the rigid surface loops. These results provide new insight into the elucidation of porin folding pathways and the factors that govern the overall structural stability of meningococcal proteins.Bacterial porins are integral outer membrane channel-forming proteins that function as molecular sieves by mediating the exchange of nutrients and waste products with the environment (1, 2). The majority of these porins assemble as trimers of a single polypeptide and exhibit an unusually high resistance to harsh conditions such as low pH, elevated temperatures, and the presence of detergents. As a consequence of such properties, porin oligomeric structures may be visualized in SDS-PAGE.1 Elucidation of the crystal structures of bacterial porins from Rhodobacter capsulatus (3), Rhodopseudomonas blastica (4), and the Escherichia coli porins OmpF, PhoE (5), and LamB (6) reveals the characteristic trimeric structure, each subunit consisting of 16 -18 antiparallel ␤-strands. Recent studies employing circular dichroism spectroscopy indicate that the meningococcal PorB class 2 protein retains physicochemical properties similar to other porins, including the predominance of ␤-sheet structure and retention of native trimeric conformation at high temperature or increased concentrations of denaturants (7).Meningococcal class 2 and class 3 proteins are gene products of two alleles of the single-copy porB gene locus (8) and are thus mutually exclusive within different Neisseria meningitidis strains (9, 10). Among the various class 2 and class 3 proteins sequenced to date, similarities in primary structure are on the order of 60 -70%. The complete amino acid sequences of N. meningitidis PorB class 3 proteins der...

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Section: Sds-induced Conformational Changes Of Classmentioning

confidence: 41%

Characterization of the Structure, Function, and Conformational Stability of PorB Class 3 Protein from Neisseria meningitidis

Minetti¹,

Blake²,

Remeta³

1998

Journal of Biological Chemistry

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“…7 However, hGH was expressed as inclusion body in E. coli, which was a major hurdle. Nevertheless, optimization of solubilization of rhGH inclusion body from E. coli was reported by Patra et al 8 Heterologous protein in periplasmic space benefits from protein folding, 9 which makes it convenient to purify rhGH. The inclusion body solubilization is unnecessary when heterologous protein is secreted in soluble form.…”

Section: Introductionmentioning

confidence: 99%

High purity recombinant human growth hormone (rhGH) expression in Escherichia coli under phoA promoter

et al. 2016

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ABSTACTRecombinant human Growth Hormone (rhGH) is an important protein for human growth and is in high demand in clinics. Hence, it is necessary to develop an efficient fermentation process to produce highly pure rhGH. In this study, rhGH was expressed in Escherichia coli under alkaline phosphatase (phoA) promoter. The cultivation conditions for high expression level and purity of rhGH were investigated. The best initial phosphate concentration for rhGH expression, out of the 4 levels of initial phosphate concentration tests performed, was 12.6 mmol/L. Subsequently, 2 fed-batch cultivations under low dissolved oxygen (DO) (0% -10%) and high DO (20% -30%) conditions were carried out. High purity rhGH (92%) was obtained from 20% -30% DO-stat cultivation, although the biomass did not show any significant difference. In summary, this research provided an efficient fermentation process for high purity rhGH production from E. coli under phoA promoter, which can lower the production and purification costs for large-scale production of rhGH.

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“…Bacterium Escherichia coli has a series of disulfide bond formation factors (Dsb) 1 in the periplasmic space-plasma membrane region of the cell (1)(2)(3). DsbA is a periplasmic enzyme having a thioredoxin-like fold and is capable of donating its Cys 30 -Cys 33 disulfide to newly translocated substrate proteins (4 -7).…”

mentioning

confidence: 99%

“…: 81-75-751-4015; Fax: 81-75-771-5699; E-mail: kito@virus.kyoto-u.ac.jp. 1 The abbreviations used are: Dsb, disulfide bond formation factors; UQ, ubiquinone; UQ1, ubiquinone-1 (a commercial chemical product used as an exogenous source of ubiquinone in vitro); DDM, n-dodecyl-␤-D-maltoside; AMS, 4-acetamido-4Ј-maleimidylstilbene-2,2Ј-disulfonate; DsbB(⌬UQ), DsbB preparation devoid of endogenously bound ubiquinone. different levels of controversy, and their unified understanding is still awaited.…”

mentioning

confidence: 99%

DsbB Elicits a Red-shift of Bound Ubiquinone during the Catalysis of DsbA Oxidation

Inaba

Takahashi

Ito

2004

Journal of Biological Chemistry

102

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DsbB is an Escherichia coli plasma membrane protein that reoxidizes the Cys 30 -Pro-His-Cys 33 active site of DsbA, the primary dithiol oxidant in the periplasm. Here we describe a novel activity of DsbB to induce an electronic transition of the bound ubiquinone molecule. This transition was characterized by a striking emergence of an absorbance peak at 500 nm giving rise to a visible pink color. The ubiquinone red-shift was observed stably for the DsbA(C33S)-DsbB complex as well as transiently by stopped flow rapid scanning spectroscopy during the reaction between wild-type DsbA and DsbB. Mutation and reconstitution experiments established that the unpaired Cys at position 44 of DsbB is primarily responsible for the chromogenic transition of ubiquinone, and this property correlates with the functional arrangement of amino acid residues in the neighborhood of Cys 44 . We propose that the Cys 44 -induced anomaly in ubiquinone represents its activated state, which drives the DsbB-mediated electron transfer.Oxidative folding is crucial for the maturation of extracytoplasmic domains of membrane and secreted proteins. Bacterium Escherichia coli has a series of disulfide bond formation factors (Dsb) 1 in the periplasmic space-plasma membrane region of the cell (1-3). DsbA is a periplasmic enzyme having a thioredoxin-like fold and is capable of donating its Cys 30 -Cys 33 disulfide to newly translocated substrate proteins (4 -7). Then, the two active site cysteines must be reoxidized for the continuing catalysis, and a plasma membrane protein, DsbB, is responsible for this reoxidation (8 -10). In vivo studies showed that the respiratory components of the cell are required for the maintenance of the oxidized state of DsbB, thus providing an oxidizing equivalent to the DsbA/DsbB system (11,12). In vitro studies showed that UQ directly activates DsbB for the DsbAoxidizing activity (13,14 UQ (12,19,20). However, recent studies suggest that the above-mentioned serial arrangement is too simple to account for the actual molecular mechanism of the DsbB-mediated DsbA oxidation reaction. Several questions have been asked. First, are the sequence of reactions driven simply by the intrinsic redox potential differences of the cysteine pairs involved? The experimental results of Inaba and Ito (20) as well as those of Regeimbal and Bardwell (21) gave a negative answer to this question. Specifically, the redox potential of Cys 104 -Cys 130 in DsbB (Ϫ250 mV) is much lower than that of Cys 30 -Cys 33 in DsbA (Ϫ120 mV) such that their in vitro reactions proceed only in the "reverse" direction. The above authors also showed that redox potential of the Cys 41 -Cys 44 pair is again lower (Ϫ210 to Ϫ270 mV) than that of DsbA. More recently, however, Grauschopf et al. (22) reached the opposite conclusion for the Cys 41 -Cys 44 redox potential (estimated to be Ϫ70 mV), using DsbB preparations chemically deprived of UQ. The second question is whether or not the reaction proceeds by sequential rearrangements of inter-and intramolecular disulf...

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Protein folding in the bacterial periplasm

Cited by 240 publications

References 87 publications

Characterization of the Structure, Function, and Conformational Stability of PorB Class 3 Protein from Neisseria meningitidis

Characterization of the Structure, Function, and Conformational Stability of PorB Class 3 Protein from Neisseria meningitidis

High purity recombinant human growth hormone (rhGH) expression in Escherichia coli under phoA promoter

DsbB Elicits a Red-shift of Bound Ubiquinone during the Catalysis of DsbA Oxidation

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